Method of obtaining viable small tissue particles and use for tissue repair

ABSTRACT

The invention provides a composition including isolated small living tissue particles, a method of making the tissue particles, and a method of using the composition to ameliorate a tissue defect. The tissue particles are composed of cells and their associated extracellular molecules and are sized, in certain embodiments, to be smaller than about 1 mm. Another aspect of the inventive tissue particles is the large percentage of viable cells. In certain embodiments, the tissue particles are made from cartilage and the composition may also contain additives such as adhesives, solutions, and bioactive agents.

TECHNICAL FIELD OF THE INVENTION

Preparation and use of tissue particles, sized from various sources, torepair tissue defects such as orthopedic tissue defects.

BACKGROUND

Articular cartilage is a thin, smooth, low friction, gliding surfacecomposed of hyaline cartilage with resiliency to compressive forces.While only a few millimeters thick, it has excellent wearcharacteristics. Its mechanical and structural capacity depends on theintegrity of its extracellular matrix, in which chondrocytes aresparsely distributed throughout structural macromolecules includingcollagen, proteoglycans, and noncollagenous proteins. Althoughchondrocyte cells produce the extracellular matrix, they compose lessthan 5% of the wet weight of cartilage.

The composition and highly complicated interaction of these componentsmake regeneration and replacement techniques challenging. For example,the lack of a direct blood supply and few cells distributed widely amonga dense extracellular matrix leads to a limited healing ability ofdamaged articular cartilage. This has led to a wide variety of treatmentapproaches for defects, for example, in the knee, with varying levels ofsuccess.

Procedures such as drilling, abrasion, microfracture, and debridementprovide symptomatic pain relief and improved function. Collectively,these procedures may be referred to as subchondral bone marrowstimulation techniques where the bone underlying the cartilage, whichhas a rich blood supply, is caused to bleed. The goal of such proceduresis to mobilize mesenchymal stem cells from the blood to differentiateinto chondrocyte-like cells that synthesize repair tissue. Once thevascularized cancellous bone is disrupted, a fibrin clot forms andpluripotent cells migrate into the area. These cells eventuallydifferentiate into chondrocyte-like cells that secrete type I, type IIand other collagen types, as well as cartilage specific proteoglycans,after receiving appropriate mechanical and biological cues. The cellsproduce a fibroblastic repair tissue that on appearance and initialbiopsy can have a hyaline-like quality, but over time, is demonstratedhistologically as being predominantly fibrocartilaginous tissue.Fibrocartilage is a relatively disorganized lattice of collagen fibers,as opposed to the natural hyaline cartilage, and thus partially fillsthe defect with structurally weak tissue that also exhibits limiteddurability.

Other procedural options such as periosteal grafting, osteochondralautografts and allografts, and autogenous chondrocyte cell implantationhave been used to repair cartilage defects for the purpose of reducingpain and restoring function. The success of these procedures generallydiminishes over time, possibly due to formation of fibrocartilage,inadequate development of repair tissue, poor cell differentiation,and/or poor bonding to the surrounding articular cartilage borders.Intact full thickness grafts, such as osteochondral autografts andallografts, also may suffer from mismatched sizes, immunologicrejection, and poor adhesion of cartilage to bony surfaces. Forautogenous chondrocyte cell implantation, two surgeries are required:chondrocytes are first obtained from an uninvolved area of cartilage andcultured for 14 to 21 days, then the cultured cells are injected intothe defect exposed via an open incision and covered with a periostealflap excised from the proximal medial tibia.

Various methods of promoting tissue growth and repair, and in particularcartilage repair, have been suggested and include the use of tissueparticles derived from grinding non-demineralized, articular cartilageinto pieces of about 60 μm to about 500 μm (Malinin U.S. PatentApplication No. 20050196460); mincing tissue into particles using twoparallel blades, resulting in particles of about 0.1 to about 3 mm³ insize and containing at least one viable cell (Binette et al. U.S. PatentApplication No. 20040078090); pulverizing soft tissue into morsels ofabout 1 to about 100 μm that may then be combined with viable elements(cells) and/or bioactive molecules (Awad et al. U.S. Patent ApplicationNo. 20050288796); and, milling allograft cartilage, which is thenlyophilized to create particles in the size of about 0.01 mm to about 1mm that can be formulated into a paste (Gomes et al. U.S. PatentApplication No. 20040219182). Various methods of tissue preparation havealso been disclosed including a method of generating dermal tissuepieces of about 50 μm to about 1500 μm using a roller with multipleblades (Mishra et al. U.S. Patent Application No. 20040175690).

Cell and/or tissue viability for implants needs to be improved. Forexample, homogenizers used to generate tissue particles have resulted inabout 5% of the cells remaining viable following homogenization.Enzymatic digestion, which is often used to generate cells forautogenous chondrocyte cell transplantation, results in poor cellviability following initial isolation.

Improved compositions and methods for repairing tissue defects and inparticular, articular cartilage defects are desired.

SUMMARY OF THE INVENTION

One embodiment is a composition including isolated small tissueparticles composed of cells and their associated extracellular molecules(e.g., proteins, polysaccharides, proteoglycans, etc.) known as theextracellular matrix (ECM). The tissue particles are sized such that insome embodiments, the particles have at least one dimension less thanabout 60 μm. In another embodiment, the particles have at least onedimension less than about 1 mm. In another embodiment, the particles aresized so that the volume is less than about 1 mm³. In some embodiments,at least about 50% of the cells in the tissue particles are viable. Inother embodiments, at least about 80% of the cells in the tissueparticles are viable. In some embodiments, the composition may alsocontain additives such as adhesives, solutions, and bioactive agents.Examples of adhesives include fibrin glue, Tisseal (Baxter BioScience,Deerfield Ill.), and Surgicel (Johnson & Johnson, New Brunswick N.J.).Examples of bioactive agents include fibrinogen, thrombin, bonemorphogenic proteins (BMP), insulin-like growth factors (IGF),transforming growth factors (TGF) including the beta form (TGF(3),platelet-derived growth factor (PDGF), and bone marrow aspirate.

Another embodiment is a method for creating small tissue particleswhereby a tissue sample is positioned on a cutting device containing atleast two blades in parallel in one embodiment, and at least threeblades in parallel in another embodiment. In embodiments containing atleast three blades, spacing between the blades may be uniform or mayvary. The space between the blades may define a dimension of theparticle. In one embodiment, at least one blade is curved. In anotherembodiment, at least two blades are not parallel. In one embodiment, thesizing apparatus comprises three blades mounted in parallel andseparated by spacers having a width of about 60 μm. By changing therelative spatial relationship between the tissue sample and the cuttingapparatus, cuts can be made in the horizontal, vertical, and coronalplanes. Because the angle between these planes can be varied, theresulting tissue particle can be sized to a variety of shapes, includingcubes, triangles, quadrilaterals, and other polygons.

Another embodiment is a method using the described compositions inameliorating a tissue defect. In one embodiment, the defective tissuemay be cartilage, bone, ligament, meniscus, tendon, muscle, nucleuspulposus, gingiva, annulus fibrosus, periosteum, perichondrium, fascia,and/or perineurium. In one embodiment, defects within articularcartilage are subjected to the method. In general, the method includesplacing the isolated sized tissue particles into a tissue defect site.Retention of the tissue particles in the defect site is facilitated bythe small particle size. In certain embodiments, retention of the tissueparticles at the defect site may be enhanced by techniques such asmicrofracture and use of adhesives.

Another embodiment is a use of the inventive small tissue particlesunder cell culture conditions and, for example, as part of in vitroexperimentation and/or to propagate cells in culture.

The method and composition will be further appreciated with reference tothe following figures and description.

BRIEF DESCRIPTION OF THE DRAWINGS

This application contains at least one drawing executed in color. APetition under 37 C.F.R. §1.84 requesting acceptance of the colordrawings is filed separately on even date herewith. Copies of thispatent or patent application publication with color drawing(s) will beprovided by the Office upon request and payment of the necessary fee.

FIG. 1A is a photograph of particles with only live cell staining.

FIG. 1B is a photograph of particles with only dead cell staining.

FIG. 2 shows an apparatus for sizing tissue particles.

FIG. 3A shows the percentage of viable cells following one embodiment ofthe method.

FIG. 3B shows the percentage of viable cells following enzymaticdigestion versus an embodiment of the invention.

FIG. 4 shows a schematic representation of the three particle planesdefined by the x-axis, y-axis, and z-axis.

FIG. 5A shows a surface of a porcine knee joint that has been subjectedto one embodiment of the invention.

FIG. 5B shows a surface of a porcine knee joint that has been subjectedto another embodiment of the invention.

DETAILED DESCRIPTION

In one embodiment, a composition comprising a plurality of isolatedtissue particles is disclosed. The particles comprise cells and theirassociated extracellular molecules, (e.g. proteins, polysaccharides,proteoglycans, etc.), which collectively are termed a matrix. In anotherembodiment, the tissue particles are comprised of cells wherein at leastabout 50% of the cells are viable. In another embodiment, the tissueparticles are comprised of cells wherein at least about 60% of the cellsare viable. In another embodiment, the tissue particles are comprised ofcells wherein at least about 65% of the cells are viable. In anotherembodiment, the tissue particles are comprised of cells wherein at leastabout 70% of the cells are viable. In another embodiment, the tissueparticles are comprised of cells wherein at least about 75% of the cellsare viable. In another embodiment, the tissue particles are comprised ofcells wherein at least about 80% of the cells are viable. Cell viabilityindicates that the cell is alive and able to perform one or moreintrinsic biological functions (e.g., cellular signaling, maintenance ofcellular homeostasis, etc.), and also may include cells that are dormantor arrested in a stage of the cell cycle. Cells that are not viable arecells that are dead. The absolute number of viable cells may varydepending on, for example, the tissue type used to make the particlesand/or particle size. The presence of viable cells in the tissueparticle composition facilitates use of the composition in amelioratingtissue defects, as described more fully below. For example, viable cellsprovide stimulators and/or cues for tissue remodeling, growth, and/orrepair. Methods to measure cell viability are known to one skilled inthe art and include facilitated dyes and biochemical assays. Forexample, cell viability in the tissue particles was measured using theLIVE/DEAD® viability assay (Invitrogen, Eugene Oreg.) where the calceindye is retained in live cells and emits a green fluorescence and theethidium homodimer is able to enter cells with damaged membrane andemits red fluorescence when interacting with nucleic acids. FIG. 1Ashows dead cells that emit red fluorescence in tissue particles in oneembodiment of the invention. FIG. 1B shows living cells that emit greenfluorescence in tissue particles in one embodiment of the invention. Theresults in FIGS. 1A and 1B showed satisfactory uptake of the dyes in tothe small tissue particles.

Along with cells, the tissue particles also contain extracellularmolecules, often referred to as the extracellular matrix (ECM). The ECMsurrounds and supports cells within mammalian tissues, and is composedof three major classes of biomolecules: (i) structural proteins such ascollagen and elastin; (ii) specialized proteins such as fibrillin,fibronectin, and laminin; and (iii) proteoglycans. Proteoglycans arecomposed of a protein core that is attached to long chains of repeatingdisaccharide units termed glycosaminoglycans (GAGs) and form complexhigh molecular weight components of the ECM. The ECM has many rolesincluding cellular organization, guidance of cell migration and growth,and structure; the prominence of these roles can vary depending on thetissue. For example, the ECM plays an important role in forcetransmission and tissue structure maintenance especially in cartilage,tendons, ligaments, bone, and muscle. The precise composition of the ECMin the particles depends on factors such as the tissue from which theparticles are obtained and any treatments or modifications thereof.Thus, the composition of the ECM will vary depending on the endogenouscomposition for that tissue type. Along with variations in ECMcomposition based on tissue type, the ECM may also be modified. As oneexample, the particles may be treated with bioactive proteins, such asBMP, IGF, TGF, PDGF, bone marrow aspirate, etc., to enhance the tissuerepair ability of the particles. Also, the particles may be treated withenzymes that hydrolyze protein and/or glycans, such as trypsin andhyaluronidase, to increase the accessability of the ECM.

The size of the tissue particles of the inventive composition may varydepending on such factors as the type of source tissue used, the age ofthe tissue, and the intended subsequent use of the composition. In oneembodiment, tissue particles are sized such that at least one dimensionof the particle is less than 1 mm. In another embodiment, the tissueparticles are sized such that at least one dimension is less than 60 μm.In another embodiment, the tissue particles are sized such that theparticle is substantially cubical with each side about 60 μm or less. Inanother embodiment, the tissue is derived from a juvenile source and thetissue particles have a volume less than 1 mm³. In another embodiment,the tissue particles have a volume of about 2×10⁻⁴ mm³. The tissueparticles may be any shape, including but not limited to cubes andelongated strips. Sizing refers to cutting of the tissue sample into thedesired size and/or shape, and is further described below.

The tissue particles may be derived from a variety of tissue types andtissue sources. The tissue may be autogenic, allogenic, or xenogenicwith respect to the recipient of the inventive composition, as explainedbelow. Any tissue is potentially suitable for use and tissue types mayinclude cartilage, bone, ligament, meniscus, tendon, muscle, nucleuspulposus, gingival, annulus fibrosus, periosteum, perichondrium, fascia,and/or perineurium. In one embodiment, the tissue is articularcartilage. In another embodiment, the articular cartilage is hyalinecartilage and/or fibrocartilage.

In one embodiment, the tissue is engineered tissue. Engineering of thetissue refers to altering the physiology of the tissue such that itpossesses traits that it would normally not have, magnifying and/ormuting the existing tissue traits, and/or growing tissue in vitro.Engineered tissue may include tissue derived from a transgenic donor.Transgenic donor refers to tissue sources, such as animals, in whichexogenous genetic material has been incorporated into the genome of thesource. The incorporated genetic material may provide for the expressionof a non-endogenous gene or may alter the expression levels of anendogenous gene. In another embodiment, the donor tissue may begenetically altered following removal from the donor. Examples ofalterations of the tissue following excision and prior to or concomitantwith culturing include alterations brought about by introduction ofgenetic material and/or bioactive agents. In the case of geneticmanipulation, the tissue may be treated with genetic vectors usingvarious methods of genetic introduction, e.g. viral- and lipid-mediated,as known in the art, to bring about alterations in endogenous orexogenous gene expression. Bioactive agents, such as growth factors, maybe incubated with the cultured tissue to bring about alterations intissue physiology. Engineered tissue may also refer to tissue that hasbeen propagated or grown in vitro. Tissue grown in vitro refers to thecreation and/or propagation of tissue outside an animal host. Forexample, in vitro grown tissue may result from tissue culturemanipulations where cells are, for example, stimulated to form a tissuein an incubating vessel. Methods for producing in vitro tissue are knownto one skilled in the art.

The developmental or maturation stage of the tissue used in theinvention may also vary. For example, the tissue particles may bederived from embryonic, fetal, neonatal, juvenile, or adult tissue. Inan embodiment where juvenile tissue is used, juvenile is defined asbeing less than 12 years old in the case of humans. Further, the tissuemay be acutely isolated or cultured prior to sizing into particles. Inthe case of cultured tissue samples, the tissue is maintained in anenvironment that preserves the viability of the cells in the tissue.However, it is also understood by one skilled in the art that some celldeath may occur as a result of in vitro tissue culturing. The tissue,either in preparation of culturing or following acute isolation, may besized into smaller pieces that either facilitate subsequent sizing, e.g.results in a size that is easier to manipulate in the subsequentcreation of tissue particles, or promotes cell viability in tissueculture, e.g. increases the surface area of the tissue and thus oxygenand nutrient accessibility to the cells. In another embodiment, thetissue is sized to the desired particle size prior to culture.

The composition may also include additional components. In oneembodiment, the tissue particles of the composition are maintained orsuspended in a solution. The solution may be a buffer that maintains thesolution pH in a desired range. For example, the buffer may maintain thetissue particles in a solution in the range from about pH 6.8 to aboutpH 7.5. In other embodiments, the buffer may maintain the pH in therange of about pH 5 to about pH 7. The buffer, and the resultingbuffering pH range chosen, depends on factors known to one skilled inthe art including the tissue type and the effects of certain pH on thattissue type.

In another embodiment, the composition include bioactive agents. Thebioactive agents may be either residual from culturing of the tissuesample as described above or may be added to the tissue particles atanother time. Examples of bioactive agents include but are not limitedto growth factors, hormones, and nutrients.

The inventive composition may also comprise an adhesive that aids in theattachment of the tissue particles to the site of tissue defect. Theadhesive may be a naturally occurring bioadhesive such as fibrin.Thrombin converts soluble plasma fibrinogen into molecules of fibrinthat polymerize and form a fibrin clot. Fibrin may encapsulate and/orenmesh the tissue particles at the sites of tissue defect. It shouldalso be noted, however, that due to the small size of the inventivetissue particles, the particles are intrinsically adhesive to the siteof tissue defect. In another embodiment, the tissue particles may betreated such that they become positively charged. The tissue particlemay be charged by a variety of treatments including exposing theparticles to an ionic detergent or a magnetic field, resulting in thecreation of an overall positive charge on the particles. The overallpositive charge of the particle facilitates adhesion of the particle tothe predominantly negatively charged tissue defect. Increased adhesionof the particles to the tissue defect site may reduce the time requiredfor tissue defect repair.

In one embodiment, a method of preparing a composition comprising tissuesized into particles is disclosed. The tissue sample is initially cutinto smaller pieces to facilitate subsequent sizing into tissueparticles, e.g., using surgical tools known to one skilled in the art,such as a scalpel. In one embodiment, the tissue is initially cut intopieces of about 5 mm to about 11 mm. In another embodiment, the tissue,which may have been cultured, has already been subjected to the initialcutting process and is of the approximate size for subsequent sizing. Asshown in the schematic of FIG. 2 (not to scale), once the tissue 10 isof the appropriate initial size, it is mounted on a jaw 12 of an axialcylinder. By extending the axial cylinder along axis A, the tissue 10contacts the blades 14 mounted in opposition of the jaw. In oneembodiment, the tissue is contacted with at least two blades mounted inparallel on a substantially flat surface. In another embodiment, thetissue is contacted with three blades mounted in parallel on asubstantially flat surface. In another embodiment, the blades 14 are notparallel to each other and may also include blades that are notstraight, e.g., curved. The configuration of blades 14 will also includespacers 16 between the blades, the width of which will correspond to thedesired dimension between parallel cuts. In certain embodiments, thespacers 16 between the blades 14 will be the same size and in otherembodiments, the spacers 16 may be of different sizes. The blades willbe sufficiently sharp so that damage and/or loss of the tissue will beminimized.

The method may be conducted in the absence of exogenously addeddigestive enzymes. Although digestive enzymes promote cell dissociation,they also may decrease the percent of viable cells resulting from thetreatment. In FIG. 3, the viability of cells following one embodiment ofthe inventive method, as determined by LIVE/DEAD® viability assay, wasabout 85% (FIG. 3A) while viability of cells following enzymatictreatment with collagenase resulted in about 25% viable cells (FIG. 3B).Without being held to a single theory, it is believed that the digestiveenzyme damages the cell membrane components, contributing to the deathof the cell.

The first contact between the blades and the tissue results in parallelcuts in, for example, the x-axis plane of the tissue sample, as shown inFIG. 4. In different embodiments, the tissue may be pushed against theblades or alternatively the blades may be pushed against the tissue. Inanother embodiment, the blade and tissue sample are both moved towardseach other. In certain embodiments, the blades may not be parallel andtherefore, would not result in parallel cuts. However, for simplicity,the inventive method will be described in terms of parallel bladesmaking parallel cuts but in all cases, the blades and resulting cuts maynot be parallel, and also may be non-straight, e.g., curved. Followingthe first contact between the blades and the tissue, the sample and/orblades can then be translationally moved along axis B, e.g. withoutrotation, so that further cuts can be made in parallel with the previouscuts and still in the same plane. In one embodiment, the blades aretranslationally moved with precision of about 1 μm using a digitalmicrometer 18, as shown in FIG. 2. The blades and/or tissue sample isthen rotated relative to one another in rotation C about axis A,defining a second orientation and the blades and tissue are again causedto contact, making cuts in, for example, the y-axis plane, as shown inFIG. 4. In this second orientation, the blades and/or tissue can againbe translationally moved so that a series of parallel cuts can be made.In one embodiment, the second contact between the blades and tissueresults in substantially perpendicular cuts in the tissue wherein theangle between the first and second cut is about 90°. However, the anglebetween the first and second cuts may range from about 1° to about 179°.In one embodiment, the blades and/or the tissue sample are moved toachieve a third orientation wherein the blades cut the tissue sample in,for example, the z-axis plane plane, as shown in FIG. 4. The thirdorientation can be achieved by moving the tissue in direction D, asshown in FIG. 2. In one embodiment, the plane defined by this thirdorientation cuts the tissue substantially perpendicular to the plane ofeither the first or second cut, for example, the x-axis or y-axis plane,and results in a particle that is substantially cubicle. However, theangle between the third and either of the first or second cutting planesmay range from about 1° to about 179°. Thus, by choosing the anglesbetween the cut planes, the geometry of the resultant tissue particlecan be varied.

Tissue particles of various sizes can be made by varying the size of thespacers between the blades and the angle between the cuts. In oneembodiment, the tissue is cut using the procedure described abovewherein three blades contact the tissue, the resultant tissue particleis sized such that at least one dimension is less than 60 μm. In anotherembodiment, the tissue is cut using the procedure described abovewherein three blades contact the tissue and the tissue is derived from ajuvenile, non-dermal source, the resultant tissue particle is sized suchthat at least one dimension is less than 1 mm. In another embodiment,the resultant tissue particle size is less than 1 mm³. The methodproduces tissue particles of the desired dimensions and also maintaineda high percentage, e.g. above about 85%, viable cells (See FIG. 3A).

As described above, the tissue source used by the inventive method togenerate tissue particles may be used from any source and with any typeof tissue including autogenic, allogenic, xenogenic, cultured, andengineered tissue and of any maturation stage.

In one embodiment, a method of ameliorating a damaged tissue in a mammalis disclosed. The tissue particle composition is introduced into or inproximity to a damaged tissue under conditions sufficient to amelioratethe damaged tissue. The composition may include a plurality of tissueparticles sized from tissue. In addition, the particles may include bothcells and extracellular molecules organized in a matrix, as describedabove. In one embodiment, the damaged tissue may be articular cartilage.In the case of damaged articular cartilage, the cartilage lesion may bedebrided back to a stable base cartilage and loose or fibrillatedcartilage may be resected. In one embodiment, in the case of articularcartilage, the subchondral base is microfractured until bleeding occursfrom the subchondral bone. Microfracture entails creating a series ofsmall fractures in the bone of about 3 mm to about 4 mm in depth usingan awl. Alternatively, a drill may be used to create holes in thesubchondral bone, with care to not cause heat necrosis in the site. Thecomposition is applied into the area of and/or proximate the defect.

In one embodiment, the method of ameliorating damaged tissue alsoincludes adhering the inventive composition to the damaged tissue. Ifthe method is conducted in conjunction with microfracture, adhesiveproperties of bleeding bone secure the tissue particles in place, asshown in FIG. 5A. Specifically, the resulting blood clot from thebleeding bone serves as a biological glue that maintains the particleson the surface near the defect. Also, due to the small size of thetissue particles, the particles naturally remain in the defect, as shownin FIG. 5B, possibly as a result of surface tension. The inventivecomposition may also include adhesives such as fibrin, hyaluronic acid,fibrin glue, fibrin clot, collagen gel, alginate gel,gelatin-resorcin-formalin adhesive, mussel-based adhesive,dihydroxyphenylalanine (DOPA) based adhesive, chitosan,transglutaminase, poly(amino acid)-based adhesive, cellulose-basedadhesive, polysaccharide-based adhesive, synthetic acrylate-basedadhesives, platelet rich plasma (PRP), platelet poor plasma (PPP), clotof PRP, clot of PPP, MATRIGEL® (BD Biosciences, San Jose CA),monostearoyl glycerol co-succinate (MGSA), monostearoyl glycerolco-succinate/polyethylene glycol (MGSA/PEG) copolymers, laminin,elastin, proteoglycans, and combinations thereof.

In one embodiment, the tissue particles of the inventive composition mayhave been treated such that the particles exhibit a net charge, asdescribed above, that facilitates electrostatic adhesion to the tissuedefect. Other techniques known to one skilled in the art, such as flaps,may also be used to keep the particles in the defect site.

In one embodiment, the method is conducted using a minimally invasiveprocedure, e.g. arthroscopy. The use of a minimally invasive procedureallows a smaller incision, resulting in less pain, a shorter in-patientstay, and a faster recovery time than traditional more invasiveprocedures. The use of a minimally invasive procedure such asarthroscopy may also aid in diminishing potential post-operativecomplications such as soft tissue fibrosis.

In one embodiment, the damaged tissue may be orthopedic tissue such ascartilage, bone, ligament, meniscus, tendon, and/or other muscle. Inanother embodiment, the damaged tissue may be nucleus pulposus,gingival, annulus fibrosus, periosteum, perichondrium, fascia, and/orperineurium. In one embodiment, the damaged tissue and the tissue thatis used as the source for the inventive composition are the same tissuetype, e.g. articular cartilage. In another embodiment, the damagedtissue and the tissue that is used as the source for the inventivecomposition are different tissue types, including autogenic, allogenic,xenogenic, cultured, engineered tissue, and of any maturation stage.

One embodiment discloses a biocompatible implantable compositioncomprising a plurality of biological tissue particles sized from tissuederived from viable juvenile cartilage, wherein the particles arecomprised of chondrocytes having at least about 80% viability andextrachrondrocyte proteins, each particle less than 60 μm, and thecomposition is capable of implantation in a mammal.

In one embodiment, particulate cartilage compositions are created andused for cartilage regeneration by stimulating chondrogenesis. Articularcartilage may be obtained from the articular surfaces of joints, such asfrom distal femurs, proximal tibia, acetabul, heads of femurs, and/orheads of radii, as well as from other sites where hyaline cartilage ispresent, e.g., auricular, nasal, temporomandibular joint, and costalmargin. The cartilage may be removed, for example, with a scalpel blade,rongeur, or other surgical instrument. In one embodiment, cartilage isremoved down to subchondral bone, without removing bone. The articularcartilage may include articular hyaline cartilage and/or fibrocartilageand may comprise allogeneic and/or xenogeneic cartilage.

The following example further illustrates embodiments of the invention.

EXAMPLE

Cartilage tissue particles were assessed for cell viability andevaluation in cartilage defect repair. All procedures were conducted incompliance with relevant regulations for the use of animal tissue.Porcine knee joints were obtained from a local abattoir. A knee jointwas opened using a scalpel and the articular cartilage from the condyleload bearing area was exposed. Using a 7.5 mm diameter coring reamer, anosteochondral plug was obtained.

The osteochondral plug was mounted on the jaw of the cutting device(FIG. 2) and a series of cuts were made using three multiple blades toobtain viable small tissue particles.

Tissue particles were stained using LIVE/DEAD® stain to determine cellviability. The LIVE/DEAD® stain uses a membrane-permeant CALCEIN AM thatis cleaved by endogenous esterases in the live cells to yieldcytoplasmic green fluorescence, and the membrane-impermeant ethidiumhomodimer-1 labels nucleic acids of membrane-compromised cells, e.g.dead cells, with red fluorescence. Pictures of the stained slides wereanalyzed using NIH imaging software and the number of total and viablecells was calculated. For the enzymatic digestion method, cartilage wasshaved off the articular surface and collected in a Petri dish. Tissueweight was recorded. The cartilage tissue blocks were digested in a 1:10mass:volume ratio in 0.15% of collagenase type II for about 12-16 hoursuntil no visible fragments remained. The cell-collagenase solution wasfiltered and washed with phosphate-buffered saline. Isolated cells werecounted and viability was determined using LIVE/DEAD® staining. Thenumber of viable cells was normalized to the tissue weight andrepresented as a percentage of the absolute number of cells in a givenunit of tissue, as shown in FIG. 3A. Paired t-test statistical analysiswas performed using Sigma Stat 2.0 software.

Results showed that tissue particles had a regular geometry. Themajority of the particles were from 50 microns to 240 microns.LIVE/DEAD® staining experiments showed good tissue penetration of thedyes due to the size of the particles. Although a small percentage,about 10% to about 15%, of the tissue was lost during the cuttingprocedure, cell viability in the tissue particles was significantlyhigher than the percentage of viable cells obtained by digestion method(Compare FIG. 3A and 3B). Tissue particles seeded on the surface of thejoint remained attached to the surface against gravity for an indefiniteperiod of time as long as conditions were maintained (FIG. 5B) and theadhesion was increased when microfracture was simulated by compressingthe subchondral bone and causing to bleed (FIG. 5A).

The above results showed that small tissue living particles wereobtained from an autologous source. The results also showed that cellviability inside of the particle remained higher than 85%. It should beunderstood that the embodiments and examples described are onlyillustrative and are not limiting in any way. Therefore, variouschanges, modifications or alterations to these embodiments may be madeor resorted to without departing from the spirit of the invention andthe scope of the following claims.

What is claimed is: 1.-35. (canceled)
 36. A composition comprising:micronized cartilage particles, the particles comprising collagen, oneor more chondrocyte-associated extracellular biomolecules and one ormore bioactive agents.
 37. The composition of claim 36, wherein thecollagen is Type II collagen.
 38. The composition of claim 36, whereinthe one or more chondrocyte-associated extracellular biomoleculescomprise proteins, polysaccharides, proteoglycans or combinationsthereof.
 39. The composition of claim 36, wherein the one or morebioactive agents comprise growth factors, hormones, nutrients orcombinations thereof.
 40. The composition of claim 39, wherein thegrowth factors comprise BMP, IGF, TGF, PDGF or combinations thereof. 41.The composition of claim 40, wherein the growth factor comprises TGF.42. The composition of claim 36, wherein the cartilage particles arederived from at least one of embryonic, fetal, neonatal, juvenile oradult tissue.
 43. The composition of claim 36, wherein the cartilageparticles comprise human juvenile cartilage particles.
 44. Thecomposition of claim 43, wherein the cartilage particles compriseisolated allogenic human juvenile cartilage particles.
 45. Thecomposition of claim 43, wherein the cartilage particles are obtainedfrom a juvenile donor less than 12 years of age.
 46. The composition ofclaim 36, further comprising an adhesive.
 47. The composition of claim46, wherein the adhesive comprises fibrin, hyaluronic acid, fibrin glue,fibrin clot, collagen gel, alginate gel, gelatin-resorcin-formalinadhesive, mussel-based adhesive, dihydroxyphenylalanine (DOPA) basedadhesive, chitosan, transglutaminase, poly(amino acid)-based adhesive,cellulose-based adhesive, polysaccharide based adhesive, syntheticacrylate-based adhesives, platelet rich plasma (PRP), platelet poorplasma (PPP), clot of PRP, clot of PPP, solubilized basement membrane(MATRIGEL®), monostearoyl glycerol co-succinate (MGSA), monostearoylglycerol co-succinate/polyethylene glycol (MGSA/PEG) copolymers,laminin, elastin, proteoglycans or combinations thereof.
 48. Thecomposition of claim 47, wherein the adhesive comprises fibrin.
 49. Thecomposition of claim 36, wherein h cartilage particles have a particlesize of from about 50 μm to about 210 μm.
 50. The composition of claim36, wherein the cartilage particles have a particle size less than about60 μm.